Artículos de revistas
Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application
Fecha
2016-11Registro en:
Guerra, Luciano Lucas; Faccinetti, Natalia Ines; Trabucchi, Aldana; Rovitto, Bruno David; Sabljic, Adriana Victoria; et al.; Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application; BioMed Central; Bmc Biotechnology; 16; 1; 11-2016; 1-19
1472-6750
CONICET Digital
CONICET
Autor
Guerra, Luciano Lucas
Faccinetti, Natalia Ines
Trabucchi, Aldana
Rovitto, Bruno David
Sabljic, Adriana Victoria
Poskus, Edgardo
Iacono, Ruben Francisco
Valdez, Silvina Noemi
Resumen
Background
The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A).
Results
The main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection.
Conclusions
E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.